Blocking eIF4E-eIF4G interaction as a strategy to impair coronavirus replication

Abstract

Coronaviruses are a family of enveloped single-stranded positive-sense RNA viruses causing respiratory, enteric, and neurologic diseases in mammals and fowl. Human coronaviruses are recognized to cause up to a third of common colds and are suspected to be involved in enteric and neurologic diseases. Coronavirus replication involves the generation of nested subgenomic mRNAs (sgmRNAs) with a common capped 5' leader sequence. The translation of most of the sgmRNAs is thought to be cap dependent and displays a requirement for eukaryotic initiation factor 4F (eIF4F), a heterotrimeric complex needed for the recruitment of 40S ribosomes. We recently reported on an ultrahigh-throughput screen to discover compounds that inhibit eIF4F activity by blocking the interaction of two of its subunits (R. Cencic et al., Proc. Natl. Acad. Sci. U. S. A. 108:1046-1051, 2011). Herein we describe a molecule from this screen that prevents the interaction between eIF4E (the cap-binding protein) and eIF4G (a large scaffolding protein), inhibiting cap-dependent translation. This inhibitor significantly decreased human coronavirus 229E (HCoV-229E) replication, reducing the percentage of infected cells and intra- and extracellular infectious virus titers. Our results support the strategy of targeting the eIF4F complex to block coronavirus infection.

Fig. 1.

Inhibition of cap-dependent translation by 4E2RCat. (A) Schematic diagram illustrating the structure of 4E2RCat. An 8-point dose-response curve of 4E2RCat in a TR-FRET assay is provided to the right. (B) Inhibition of translation by 4E2RCat. Schematic representation of FF/HCV/Ren bicistronic construct used for in vitro translation studies (top). In vitro translations were performed in Krebs extracts programmed with FF/HCV/Ren in the presence of [³⁵S]methionine, and a representative autoradiograph of the products after fractionation on 10% SDS-PAGE is provided (bottom left). Translations contained vehicle (1% DMSO) (lane 1), 500 μM m⁷GDP (lane 2), 500 μM GDP (lane 3), 50 μM anisomycin (lane 4), the indicated concentrations of 4E2RCat (lanes 5 to 10), or no RNA (lane 11). FF and Ren RLU values (relative to DMSO controls) from two independent experiments are provided with the standard errors of the means (SEM) indicated (bottom right). (C) Schematic representation of FF/EMCV/Ren bicistronic construct used for in vitro translation studies (top). RLU values (relative to those of the DMSO control) from two independent in vitro translations performed in Krebs extract programmed with FF/EMCV/Ren mRNA are provided with the SEM indicated (bottom).

Fig. 2.

Inhibition of eIF4E-eIF4G and eIF4E-4E-BP1 interaction by 4E2RCat. (A) Assessing the effect of 4E2RCat on the interaction between eIF4E and its binding partners. On the left, eIF4E (lanes 1 and 2) or BSA (lane 3) and Affi-Gel-coupled GST-eIF4GI_517-606 were incubated in the presence of vehicle (1% DMSO) or 100 μM 4E2RCat, and the effects on interaction were assessed in pull-down assays as described in Materials and Methods. The gels in the middle and on the right show the consequences of 4E2RCat on eIF4E-GST-eIF4GII_555-568 and eIF4E-GST-4E-BP1 interaction. The asterisk denotes the position of the migration of the GST fusion protein. (B) Effect of 4E2RCat on eIF4F assembly. RSW was incubated with vehicle or 25 μM 4E2RCat for 1 h at 30°C, followed by pulldowns using 50 μl of 50% m⁷GTP-Sepharose beads (GE Healthcare) for 2 h end over end at 4°C. GTP and m⁷GTP eluents are presented. (C) Inhibition of translation in vivo by 4E2RCat. L132 cells were exposed to the indicated concentrations of 4E2RCat for 4 or 24 h, after which metabolic labeling was performed. Results are the averages from triplicates with the errors of the means shown, and values are standardized against total protein content. (D) 4E2RCat does not induce cell death. Fraction of apoptotic cells following exposure of L132 cells to 12.5 μM 4E2RCat for the indicated time periods. Samples were prepared as described in Materials and Methods, and flow cytometry was performed to determine the fraction of apoptotic cells relative to the value for the DMSO vehicle control, which was set to 1.

Fig. 3.

SAR and in silico analysis of 4E2RCat. (A) Chemical structures of 4 of the 19 most potent congeners tested that inhibited cap-dependent translation in vitro. (B) Affi-Gel pulldown experiments with GST-eIF4GI_517-606 and eIF4E in the presence of either DMSO (1%) or the indicated compounds at a final concentration of 100 μM. (C) Inhibition of in vivo protein synthesis of analogs of 4E2RCat. MDA-MB-231 cells were treated for 4 h with the indicated compounds at a concentration of 25 μM, after which metabolic labeling was performed. Results are the averages from duplicates with the errors of the mean shown, and values are standardized against total protein content. (D) Location of the largest hot spots of eIF4E. The site (shown in yellow) binds 24 probe clusters and defines the main hot spot. The other large consensus sites are shown in magenta (22 probe clusters), cyan (19 probe clusters), and salmon (10 probe clusters). A small consensus site is shown in ochre (5 probe clusters) and indicates a shallow channel connecting two other consensus sites. (E) The most likely binding pose of 4E2RCat. The predicted hot spots are superimposed for reference.

Fig. 4.

Inhibition of coronavirus replication by 4E2RCat. (A) Coronavirus replication is eIF4F dependent. Following infection and exposure to either vehicle (0.2% DMSO) or the indicated concentrations of hippuristanol, silvestrol, 4E1RCat, or 4E2RCat, infectious viral titers released from the cells (extracellular) were determined, and the averages from three independent experiments are presented. Error bars denote standard errors of the means. LOD, limit of detection (denoted by the red dashed line). (B) Effects of eIF4A and eIF4E-eIF4G inhibition on intracellular infectious virus production. (C) Effects of eIF4A and eIF4E-eIF4G inhibition on the production of HCoV-229E S protein. Following infection and exposure to vehicle or compounds for 24 h, infected cells were processed for immunofluorescence using a mouse IgG1 MAb, 5-11H.6, followed by AlexaFluor-488 anti-mouse goat antibody (green). Nuclei are stained with DAPI (blue). (D) Percent S protein-positive cells in HCoV-229E-infected cells.

Fig. 5.

Effects of 4E2RCat on coronavirus infection. (A) 4E2RCat inhibits coronavirus replication in a dose- and time-dependent manner. Following the exposure of HCoV-229E-infected L132 cells to the indicated concentrations of 4E2RCat for the indicated periods of time, infectious viral titers released from the cells (extracellular) or from intracellular infectious virus were determined, and the averages from three independent experiments are presented. Error bars denote SEM. The limit of detection is denoted by the red dashed line. (B) Dose-dependent inhibition of HCoV-229E S protein production by 4E2RCat. Following infection and exposure to vehicle or the indicated concentrations of 4E2RCat for 24 h, infected cells were processed for immunofluorescence using mouse IgG1 MAb 5-11H.6 followed by AlexaFluor-488 anti-mouse goat antibody (green). Nuclei are stained with DAPI (blue). (C) Metabolic labeling of HeLa cells infected with type 1 poliovirus (Mahoney) followed by treatment with vehicle (1% DMSO) or 50 μM 4E2RCat for 4 h.